Anti-rat kallistatin antibody expressed in mouse hybridoma cells was generated through a contracted service at Proteintech (Rosemont, IL). The rat and mouse eyeballs were fixed in Davidson’s fixation solution for 48 h for the paraffin section. Following the antigen retrieval with sodium citrate buffer (10 mmol/L sodium citrate, 0.05% Tween 20, pH 6.0) in steam bath and blocking, the sections were incubated with anti-rat kallistatin antibody or anti-nonphosphorylated β-catenin antibody (Cell Signaling, no. 8814) overnight. After being washed with PBS, the slides were incubated with A488-labeled goat anti-mouse IgG (Jackson ImmunoResearch, no. 115-545-003) or A488-labeled goat anti-rabbit IgG (Jackson ImmunoResearch, no. 111-545-003). The slides were mounted with Vectashield mounting buffer containing DAPI (Vector Laboratories, no. H-1200) and photographed under a Zeiss Microscope (Observer Z1, Pleasanton, CA).
Anti nonphosphorylated β catenin antibody
Manufactured by Cell Signaling Technology
Sourced in United States
The Anti-nonphosphorylated β-catenin antibody is a laboratory tool used to detect the non-phosphorylated form of the β-catenin protein. β-catenin is a key component of the Wnt signaling pathway and plays a crucial role in various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Anti egfr antibody, by Abcam (1 mentions)
Mouse anti β actin antibody, by Merck Group (1 mentions)
Horseradish peroxidase labeled secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Gapdh, by Abcam (1 mentions)
Proteinase k, by Qiagen (1 mentions)
Bioruptor plus sonication device, by Diagenode (1 mentions)
3 protocols using anti nonphosphorylated β catenin antibody
1
Immunohistochemical Analysis of Kallistatin and β-Catenin in Rat and Mouse Eyeballs
2
Kallistatin Protein Expression Analysis
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Western blot analysis was performed as described previously (32 (link)). Anti-human kallistatin monoclonal antibodies were gifts from Drs. L. Chao and J. Chao (21 (link),22 (link)). GAPDH (Abcam, no. ab9485), rabbit anti-kallistatin antibody (Abcam, no. ab187656), anti-nonphosphorylated β-catenin antibody (Cell Signaling. no. 8814), anti-EGFR antibody (Abcam, no. ab52894), and mouse anti–β-actin antibody (Sigma-Aldrich, no. A5441) were used as primary antibodies at 1:1,000 dilution. Horseradish peroxidase-labeled secondary antibodies (12,000 dilution; Santa Cruz Biotechnology) were used for immunoblotting. Densitometry was performed using ImageJ software and normalized by β-actin levels.
3
Mapping β-catenin Binding Sites in SCN9A
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The 17-week-old MPS II neurons were first crosslinked with 1% formaldehyde solution, followed by reverse crosslinking with 0.125 M glycine solution. The cells were then lysed on ice for 15 min in ChIP lysis buffer and sonicated for 30 sec on and 30 sec off in five cycles using a Bioruptor Plus sonication device (Diagenode, NJ, USA). The isolated 200–1000 base pairs of DNA fragments were immunoprecipitated with anti-non-phosphorylated β-catenin antibody (Cell Signaling, MA, USA) at 4 °C overnight, followed by incubation with 50 µl of protein G (Cytiva, MA, USA) at 4 °C for 4 h. The recovery of DNA fragments was achieved by reversing the crosslinking process using 5 mM NaCl and 40 µg RNaseA at 65 °C overnight, followed by 4 µl (10 mg/ml) of proteinase K (Qiagen Inc., CA, USA) at 60 °C for 1 h. The immunocomplex was purified by ethanol precipitation after being washed and eluted from beads. The human SCN9A promoter region was detected using PCR and quantified using qRT-PCR.
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